Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase

Citation
Y. Nakanishi et al., Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase, J BIOL CHEM, 276(10), 2001, pp. 7654-7660
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
276
Issue
10
Year of publication
2001
Pages
7654 - 7660
Database
ISI
SICI code
0021-9258(20010309)276:10<7654:MAOFRI>2.0.ZU;2-D
Abstract
Vacuolar H+-translocating inorganic pyrophosphatase(V-PPase) uses PP, as an energy donor and requires, free Mg2+ for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (ASp(253), Ly s(261), GlU(263), ASp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp (731)) in the putative PPi-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conserv ative residues resulted in a marked decrease in PPi hydrolysis activity and a complete loss of H+ transport activity. The conformational change of V-P Pase induced by the binding of the substrate was reflected in the susceptib ility to trypsin, Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PPi, while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These re sults suggest that the second acidic region is also implicated in the subst rate hydrolysis and that at least two residues, Lys(261) and GLU(263), are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PPi hydrol ysis but no proton pump activity, we estimated that two residues, Lys(261) and GlU(263), might be related to the energy conversion from PP, hydrolysis to H+ transport. The importance of two residues, Asp(253) and GlU(263), in the Mg2+-binding function was also suggested from the trypsin susceptibili ty in the presence of Mg2+. Furthermore, it was found that the two acidic r egions include essential common motifs shared among the P-type ATPases.